1. Field of the Invention
This invention relates to improved composition of matter containing polyalkylene oxide and a biologically active proteinaceous substance, process of making the composition of matter and method of using the same for the treatment of disease processes associated with various physiological disorders in which the administration of the biologically active proteinaceous substance effects an immune response.
More particularly, this invention relates to improved composition of matter containing polyethylene glycol-superoxide dismutase, process of making the composition of matter and method of using the same for the treatment of disease processes associated with the adverse effects on tissue of superoxide anions, such as ischemic events, reperfusion injury, trauma and inflammation.
2 . Reported Developments
Biologically active proteins, particularly enzymes and peptide hormones, have been long considered as ideal drugs for the treatment of various diseases due to their specificity and rapid catalytic action. Such enzymes include:
Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3; "uricase"); Hydrogen-peroxide: hydrogen-peroxide oxidoreductase (1.11.1.6; "catalase"); Cholesterol, reduced--NADP: oxygen oxidoreductase (20-.beta.-hydroxylating) (1.14.1.9; "Cholesterol 20-hydroxylase"). PA1 Transferases such as: UDP glucuronate glucuronyl-transferase (acceptor unspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose: .alpha.-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12). PA1 Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17; lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1; "Asparaginase"). PA1 Lyases such as: Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase (4.1.2.12; "aldolase"). PA1 Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase) and PA1 Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5). PA1 Insulin, ACTH, Glucagon, Somatostatin, Somatotropin, Thymosin, Parathyroid hormone, Pigmentary hormones, Somatomedin, Erythropoietin, Luteinizing hormone, Chorionic Gonadotropin, Hypothalmic releasing factors, Antidiuretic hormones, Thyroid stimulating hormone, Calcitonin and Prolactin. PA1 a) LDPEG+carboxylating agent.fwdarw.LDPEG-COOH PA1 b) LDPEG-COOH+carboxyl group activating agent.fwdarw.active ester of LDPEG-COOH PA1 c) n (active esters of LDPEG-COOH)+Protein.fwdarw.(LDPEG-CO).sub.n -Protein PA1 Recombinant human interleukin-4 (rhuIL-4); PA1 Protease Subtilisin Carlsberg; PA1 Superoxide dismutases such as bovine, human, and various recombinant superoxide dismutases such as recombinant human superoxide dismutase (rhuSOD); PA1 Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3; "uricase"); Hydrogen-peroxide: hydrogen-peroxide oxidoreductase (1.11.1.6; "catalase"); Cholesterol, reduced--NADP: oxygen oxidoreductase (20-.beta.-hydroxylating) (1.14.1.9; "Cholesterol 20-hydroxylase"); PA1 Transferases such as: UDP glucuronate glucuronyl-transferase (acceptor unspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose: .alpha.-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12); PA1 Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17; lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1; "Asparaginase"); PA1 Lyases such as: Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase (4.1.2.12; "aldolase"); PA1 Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase) and PA1 Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5). PA1 Insulin, ACTH, Glucagon, Somatostatin, Somatotropin, Thymosin, Parathyroid hormone, Pigmentary hormones, Somatomedin, Erythropoietin, Luteinizing hormone, Chorionic Gonadotropin, Hypothalmic releasing factors, Antidiuretic hormones, Thyroid stimulating hormone, Calcitonin and Prolactin. PA1 LDPEG-SS=low diol CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2 COO(C.sub.4 H.sub.4 NO.sub.2) containing not more than 10% of [(C.sub.4 H.sub.4 O.sub.2 N)OOC--CH.sub.2 CH.sub.2 COO].sub.2 PEG PA1 LDPEG-S=low diol CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2 COOH containing not more than 10% of [HOOC--CH.sub.2 CH.sub.2 --COO].sub.2 PEG PA1 DCC=dicyclohexylcarbodiimide PA1 SA=succinic anhydride PA1 bSOD=Bovine Superoxide Dismutase PA1 NHS=(C.sub.4 H.sub.4 NO.sub.2)OH, N-hydroxysuccinimide PA1 (LDPEG).sub.n bSOD=low diol(CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2 CO).sub.n --bSOD PA1 (LDPEG).sub.n-l bSOD-S=low diol(CH.sub.3 O-PEG-OCOCH.sub.2 CH.sub.2 CO).sub.n-1 --bSOD-COCH.sub.2 CH.sub.2 COOH PA1 SAcid=Succinic Acid PA1 n=number of low diol PEGs per SOD PA1 K.sub.1, K.sub.obs, k.sub.2 and k.sub.3 are rate constants for the reactions.
The peptide hormones include:
However, with minor exceptions, enzyme therapy, particularly with non-human enzymes, has been less than successful due in part to the enzymes' relatively short half-lives and to their respective immunogenicities. Upon administration, the host defense system responds to remove the foreign enzymes by initiating the production of antibodies thereto, thereby substantially reducing or eliminating their therapeutic efficacies. Repeated administration of foreign and of otherwise short lived human enzymes is essentially ineffective, and can be dangerous because of concomitant allergic response. Various attempts have been taken to solve these problems, such as through microencapsulation, entrapment in liposomes, genetic engineering and attachment of the enzymes to polymers. Among the attempts the most promising appears to be the chemical attachment of the proteinaceous substances to polyalkylene oxide (PAO) polymers and particularly polyethylene glycols (PEG). The following illustrates these attempts.
U.S. Pat. No. 4,179,337 discloses the use of polyethylene glycol or polypropylene glycol coupled to proteins to provide a physiologically active non-immunogenic water soluble polypeptide composition in which the polyethylene glycol (hereinafter sometimes referred to as PEG) serves to protect the polypeptide from loss of activity without inducing substantial immunogenic response. The methods described in the patent for the coupling of polyethylene glycol to a protein involve either the conversion of a protein amino group into an amide or pseudoamide, with consequent loss of charge carrying capacity of the amino group, or the introduction at the amino group of the protein, or vicinal to it, of a heteroatom substituent such as a hydroxyl group or of a ring system that is not repeated in the polymer backbone.
Veronese, F. M., Boccu, E., Schaivon, O., Velo, G. P., Conforti, A., Franco, L., and Milanino, R., in Journal of Pharmacy and Pharmacology, 35, 757-758 (1983), reported that when bovine erythrocyte derived superoxide dismutase is modified with a polyethylene glycol carboxylic acid N-hydroxysuccinimide active ester, the half-life of the enzyme in rats is increased over that of the unmodified protein.
European Patent Application 0 200 467 of Anjinomoto, Inc. describes superoxide dismutase that is chemically modified by a polyalkylene oxide (PAO) which is functionalized at both ends of the polymer with activated carboxyl coupling groups, each capable of reacting with protein. Because the activated coupling sites are located at opposite ends of the polymer chain, it is unlikely that the presence of an activated group at one end of the polymer can have a significant effect on the reactive nature of the group at the other end of the polymer. These polymers are capable of reacting at both ends to cross-couple with proteins to form copolymers between the protein and the polyalkylene oxide. Such copolymers do not have well defined or molecularly stoichiometric compositions.
Veronese, F. M. et al in Journal of Controlled Release, 10, 145-154 (1989) report that the derivatization with monomethoxypolyethylene glycol (hereinafter sometimes referred to as MPEG) of superoxide dismutase (hereinafter sometimes referred to as SOD) gives a heterogenous mixture of products. Heterogeneity was demonstrated to depend on the presence of bifunctional polyethylene glycol (DPEG) in the monofunctional methoxylated molecules.
These attempts, in general, have resulted in longer half-life and reduced immunogenicity of the proteinaceous biologically active substances. However, further improvements are needed in order to successfully treat a variety of diseases with these promising biologicals.
We have now discovered that biologically active proteinaceous substances can be made to possess longer half-life and less immunogenic properties by chemically modifying them using low diol polyalkylene oxide, particularly low diol polyethylene glycol (hereinafter sometimes referred to as LDPEG).
The invention will be described with specific reference to SOD, however, it is to be understood that other biologically active substances may also be chemically modified using low diol polyalkylene oxides (hereinafter sometimes referred to as LDPAO).